Thymidylate synthase (TS) catalyzes the reductive methylation of dUMP to dTMP, a reaction that is essential for the de novo biosynthesis of dTTP for DNA replication and cell proliferation. Inhibitors of TS, such as the fluoropyrimidine antimetabolite 5-fluorouracil, have been important in the chemotherapy of cancer for quite some time, and remain a mainstay in the clinical management of neoplastic disease. A large body of research on TS and its inhibitors has led to detailed knowledge of the enzyme's structure (which governs its affinity for inhibitory ligands) and synthesis (which controls its intracellular concentrations). Recent studies by our group have identified new aspects of TS biology that will be important to further advances in the use of TS inhibitors for cancer treatment. The research program we propose in this application is a comprehensive one that focuses around several novel issues, including: the contribution of reactive oxygen species to the toxicity of TS inhibitors (AIM 1); post-translational modification of TS and regulation of its intracellular locale (AIM 2); and the function of nuclear TS in DNA repair and attenuation of genome damage (AIM 3). We suggest that response to TS inhibitors drugs depends upon the balance between ROS-dependent cell death and DNA repair-dependent cell survival. In all, this application proposes studies of newly recognized features of TS that should lead to novel insights into the role of this important drug target in cancer therapy. PUBLIC HEALTH RELEVANCE: Thymidylate synthase (TS) has been an important target for anticancer chemotherapy for decades. The goal of the proposed project is to test the effects of modulating the levels of reactive oxygen species (ROS) on tumor response to TS inhibitors and examine the ability of FUra to potentiate the cytotoxicity of different classes of anti-neoplastic agents. The results of these studies will be valuable in developing new and novel therapies in combination with TS inhibitors that might synergize to enhance their anti-tumor efficacy.